Rotation manipulation of single-molecule magnetic trapping and gene transcription regulation dynamics

Abstract

Gene transcription regulation is a key step for gene expression in all organisms and responsible for the transmission of genetic information and genome integrity. As one of the most important mechanisms in gene transcription, an RNA polymerase (RNAP) specifically interacts with and unwinds genome DNA to form a transcription bubble where a nascent RNA transcript is polymerized, taking one of the unwound DNA strands as its template. The RNAP translocates along the DNA to transcribe the whole gene by carrying the transcription bubble. In such a way, an RNAP completes its biological task of gene expression by physically acting as a molecular machinery. Thus, an RNAP molecule can be considered as a research object for physicists who are willing to uncover the mechanisms of life processes in a physical view. To achieve this, single-molecule method has been invented and used widely. As one of these methods, single-molecule magnetic trapping manipulates biological molecules by applying extension force or torque to the magnetic beads tethered through biological molecule to pre-coated glass surfaces by manipulating the position or rotation of a pair of magnets. A linear DNA molecule can be manipulated in such a way to generate plectonemes, i.e. DNA supercoils, under an extension force of 0.3 pN (1 pN = 10<sup>–12</sup> N), possessing the feature that the number of unwound base pairs of a supercoiled DNA can be observed by the changes in the number of supercoils reflected by the DNA extension changes. Thus, the DNA unwound by RNAP, i.e. the transcription bubble, during transcription can be observed in this way. By monitoring the kinetics of the transcription bubble in real time, this method thus allows single-molecule detection with single-base resolution and a high-throughput data collection fashion in the kinetic studies of transcription. Owing to the advantages of the manipulation of DNA supercoils with single-molecule magnetic trapping, one can mimic the mechanistic feature of DNAs in vivo and characterize the kinetics of transcription under such conditions. This method can also be combined with single-molecule fluorescence method which can be applied to studying the mechanism of transcription regulation while monitoring the behaviors of fluorescently labeled biological molecules that interact with functional RNAP molecules, providing examples for studying the mechanisms of transcription regulations in more complex systems.